A recombinant HIV-2 (NIH-Z) Nef protein synthesized in E. coli, and purified by the detergent/chaotrope extraction technique and preparative SDS-PAGE is: immunologically reactive on Western blots with anti-Nef antibodies; an excellent substrate in vitro for phosphorylation both by purified protein kinase C (PKC) and the maturation promoting factor (MPF) kinase purified from Xenopus oocytes; exhibits an intrinsic low-level autokinase activity, and forms stable homodimers and homotetrameric complexes in vitro, which are significantly increased in the absence of sulfhydryl-reducing agents. Preliminary results of in vivo phosphory- lation and oligomerization experiments suggest that the native 25 kD HIV-2 (NIH-Z) Nef protein in infected T-lymphocytes in culture becomes highly phosphorylated following stimulation with calcium ionophores, and the dimeric (50 kD) and tetrameric (100 kD) forms of this protein can be radioimmunoprecipitated with specific anti-HIV-2 Nef monoclonal antibodies. In addition, evidence of a putative Nef cellular protein complex was also obtained. Structural studies have also revealed the presence of a leucine zipper-like repeat structure at the conserved central """"""""core"""""""" region of the Nef proteins, with a characteristic 4,3 repeat similar to the heptad leucine repeat motif of the bZIP factors. Moreover, at the C-terminus of the Nef proteins is a highly acidic sequence (net charge of -5 to -8) stretched over 40 amino acids, and it contains two predicted alpha-helices separated by a predicted beta-turn structure, with homology to known acidic activation domains of transcriptional activation factors. Biochemical characterization of individual biological Nef clones expressing only p27nef (clone 3B-3) or p25nef (clone 3B-5), isolated by limiting dilution of HTLV-IIIB-infected H9 cells, are continuing. In addition, a baculovirus vector-expressed HIV-1 Vpu protein, which contains a potential calcium binding or calcium channel-like sequence similar to one of the S-IV sequences of the dihydropyridine (DHP)-receptor, is being purified for use in calcium channel drug-binding studies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01CP005120-12
Application #
3853416
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Division of Cancer Epidemiology and Genetics
Department
Type
DUNS #
City
State
Country
United States
Zip Code